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. 1977 Jun;4(6):1739–1757. doi: 10.1093/nar/4.6.1739

Synthesis of messenger RNA-like molecules in isolated myeloma nuclei.

Y Y Mory, M L Gefter
PMCID: PMC342518  PMID: 561391

Abstract

Nuclei isolated from mouse myeloma cells grown in tissue culture are capable of synthesizing RNA for prolonged periods of time. Addition of cytoplasmic extracts to the system stimulates slightly the rate and prolongs the time of synthesis. As judges by sedimentation in SDS and in formamide gradients, the size of the RNA synthesized is heterogeneous from smaller than 10S to larger than 45S, thus resembling in vivo made RNA. The characteristics of some of the RNA are in keeping with those expected to be for mRNA. Fifty percent of the RNA synthesis is sensitive to alpha-amanitin. After an incubation of two hours in the absence of alpha-amanitin about 10 percent of the newly synthesized RNA is found outside of the nuclei; it sediments with a broad distribution at 18S. A considerable fraction of the RNA that is released from nuclei in vitro can promote the formation of polyribosomes, and contains molecules that are polyadenylated and "capped".

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Selected References

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  1. Abraham G., Rhodes D. P., Banerjee A. K. The 5' terminal structure of the methylated mRNA synthesized in vitro by vesicular stomatitis virus. Cell. 1975 May;5(1):51–58. doi: 10.1016/0092-8674(75)90091-4. [DOI] [PubMed] [Google Scholar]
  2. Aviv H., Boime I., Leder P. Protein synthesis directed by encephalomyocarditis virus RNA: properties of a transfer RNA-dependent system. Proc Natl Acad Sci U S A. 1971 Sep;68(9):2303–2307. doi: 10.1073/pnas.68.9.2303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Banks S. P., Johnson T. C. Synthesis of RNA-polyadenylic acid by isolated brain nuclei. Science. 1973 Sep 14;181(4104):1064–1065. doi: 10.1126/science.181.4104.1064. [DOI] [PubMed] [Google Scholar]
  4. Cory S., Adams J. M. The modified 5'-terminal sequences in messenger RNA of mouse myeloma cells. J Mol Biol. 1975 Dec 25;99(4):519–547. doi: 10.1016/s0022-2836(75)80170-7. [DOI] [PubMed] [Google Scholar]
  5. Darnell J. E., Wall R., Tushinski R. J. An adenylic acid-rich sequence in messenger RNA of HeLa cells and its possible relationship to reiterated sites in DNA. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1321–1325. doi: 10.1073/pnas.68.6.1321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Edmonds M., Caramela M. G. The isolation and characterization of adenosine monophosphate-rich polynucleotides synthesized by Ehrlich ascites cells. J Biol Chem. 1969 Mar 10;244(5):1314–1324. [PubMed] [Google Scholar]
  7. Edmonds M., Vaughan M. H., Jr, Nakazato H. Polyadenylic acid sequences in the heterogeneous nuclear RNA and rapidly-labeled polyribosomal RNA of HeLa cells: possible evidence for a precursor relationship. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1336–1340. doi: 10.1073/pnas.68.6.1336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Furuichi Y., Morgan M., Muthukrishnan S., Shatkin A. J. Reovirus messenger RNA contains a methylated, blocked 5'-terminal structure: m-7G(5')ppp(5')G-MpCp-. Proc Natl Acad Sci U S A. 1975 Jan;72(1):362–366. doi: 10.1073/pnas.72.1.362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Furuichi Y., Morgan M., Shatkin A. J., Jelinek W., Salditt-Georgieff M., Darnell J. E. Methylated, blocked 5 termini in HeLa cell mRNA. Proc Natl Acad Sci U S A. 1975 May;72(5):1904–1908. doi: 10.1073/pnas.72.5.1904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Glisin V., Crkvenjakov R., Byus C. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry. 1974 Jun 4;13(12):2633–2637. doi: 10.1021/bi00709a025. [DOI] [PubMed] [Google Scholar]
  11. Groner Y., Hurwitz J. Synthesis of RNA containing a methylated blocked 5' terminus by HeLa nuclear homogenates. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2930–2934. doi: 10.1073/pnas.72.8.2930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jacobson A., Firtel R. A., Lodish H. F. Synthesis of messenger and ribosomal RNA precursors in isolated nuclei of the cellular slime mold Dictyostelium discoideum. J Mol Biol. 1974 Jan 15;82(2):213–230. doi: 10.1016/0022-2836(74)90342-8. [DOI] [PubMed] [Google Scholar]
  13. Lee S. Y., Mendecki J., Brawerman G. A polynucleotide segment rich in adenylic acid in the rapidly-labeled polyribosomal RNA component of mouse sarcoma 180 ascites cells. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1331–1335. doi: 10.1073/pnas.68.6.1331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lindell T. J., Weinberg F., Morris P. W., Roeder R. G., Rutter W. J. Specific inhibition of nuclear RNA polymerase II by alpha-amanitin. Science. 1970 Oct 23;170(3956):447–449. doi: 10.1126/science.170.3956.447. [DOI] [PubMed] [Google Scholar]
  15. Lodish H. F. Alpha and beta globin messenger ribonucleic acid. Different amounts and rates of initiation of translation. J Biol Chem. 1971 Dec 10;246(23):7131–7138. [PubMed] [Google Scholar]
  16. Marzluff W. F., Jr, Murphy E. C., Jr, Huang R. C. Transcription of ribonucleic acid in isolated mouse myeloma nuclei. Biochemistry. 1973 Aug 28;12(18):3440–3446. doi: 10.1021/bi00742a013. [DOI] [PubMed] [Google Scholar]
  17. Penman S., Vesco C., Penman M. Localization and kinetics of formation of nuclear heterodisperse RNA, cytoplasmic heterodisperse RNA and polyribosome-associated messenger RNA in HeLa cells. J Mol Biol. 1968 May 28;34(1):49–60. doi: 10.1016/0022-2836(68)90234-9. [DOI] [PubMed] [Google Scholar]
  18. Perry R. P., Kelley D. E., Friderici K., Rottman F. The methylated constituents of L cell messenger RNA: evidence for an unusual cluster at the 5' terminus. Cell. 1975 Apr;4(4):387–394. doi: 10.1016/0092-8674(75)90159-2. [DOI] [PubMed] [Google Scholar]
  19. Perry R. P., Kelley D. E. Messenger RNA-protein complexes and newly synthesized ribosomal subunits: analysis of free particles and components of polyribosomes. J Mol Biol. 1968 Jul 14;35(1):37–59. doi: 10.1016/s0022-2836(68)80035-x. [DOI] [PubMed] [Google Scholar]
  20. Perry R. P., Kelley D. E. Methylated constituents of heterogeneous nuclear RNA: presence in blocked 5' terminal structures. Cell. 1975 Sep;6(1):13–19. doi: 10.1016/0092-8674(75)90068-9. [DOI] [PubMed] [Google Scholar]
  21. Price R., Penman S. A distinct RNA polymerase activity, synthesizing 5-5 s, 5 s and 4 s RNA in nuclei from adenovirus 2-infected HeLa cells. J Mol Biol. 1972 Oct 14;70(3):435–450. doi: 10.1016/0022-2836(72)90551-7. [DOI] [PubMed] [Google Scholar]
  22. Price R., Penman S. Transcription of the adenovirus genome by an -amanitine-sensitive ribonucleic acid polymerase in HeLa cells. J Virol. 1972 Apr;9(4):621–626. doi: 10.1128/jvi.9.4.621-626.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Raskas H. J. Release of adenovirus messenger RNA from isolated nuclei. Nat New Biol. 1971 Sep 29;233(39):134–136. doi: 10.1038/newbio233134a0. [DOI] [PubMed] [Google Scholar]
  24. Reeder R. H., Roeder R. G. Ribosomal RNA synthesis in isolated nuclei. J Mol Biol. 1972 Jun 28;67(3):433–441. doi: 10.1016/0022-2836(72)90461-5. [DOI] [PubMed] [Google Scholar]
  25. Rose J. K. Heterogneeous 5'-terminal structures occur on vesicular stomatitis virus mRNAs. J Biol Chem. 1975 Oct 25;250(20):8098–8104. [PubMed] [Google Scholar]
  26. Salditt-Georgieff M., Jelinek W., Darnell J. E., Furuichi Y., Morgan M., Shatkin A. Methyl labeling of HeLa cell hnRNA: a comparison with mRNA. Cell. 1976 Feb;7(2):227–237. doi: 10.1016/0092-8674(76)90022-2. [DOI] [PubMed] [Google Scholar]
  27. Singer R. H., Penman S. Messenger RNA in HeLa cells: kinetics of formation and decay. J Mol Biol. 1973 Aug 5;78(2):321–334. doi: 10.1016/0022-2836(73)90119-8. [DOI] [PubMed] [Google Scholar]
  28. Villa-Komaroff L., McDowell M., Baltimore D., Lodish H. F. Translation of reovirus mRNA, poliovirus RNA and bacteriophage Qbeta RNA in cell-free extracts of mammalian cells. Methods Enzymol. 1974;30:709–723. doi: 10.1016/0076-6879(74)30068-7. [DOI] [PubMed] [Google Scholar]
  29. Wallace R. D., Kates J. State of adenovirus 2 deoxyribonucleic acid in the nucleus and its mode of transcription: studies with isolated viral deoxyribonucleic acid-protein complexes and isolated nuclei. J Virol. 1972 Apr;9(4):627–635. doi: 10.1128/jvi.9.4.627-635.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wei C. M., Gershowitz A., Moss B. Methylated nucleotides block 5' terminus of HeLa cell messenger RNA. Cell. 1975 Apr;4(4):379–386. doi: 10.1016/0092-8674(75)90158-0. [DOI] [PubMed] [Google Scholar]
  31. Wei C. M., Moss B. Methylation of newly synthesized viral messenger RNA by an enzyme in vaccinia virus. Proc Natl Acad Sci U S A. 1974 Aug;71(8):3014–3018. doi: 10.1073/pnas.71.8.3014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Winicov I., Perry R. P. Synthesis methylation, and capping of nuclear RNA by a subcellular system. Biochemistry. 1976 Nov 16;15(23):5039–5046. doi: 10.1021/bi00668a014. [DOI] [PubMed] [Google Scholar]
  33. Zylber E. A., Penman S. Products of RNA polymerases in HeLa cell nuclei. Proc Natl Acad Sci U S A. 1971 Nov;68(11):2861–2865. doi: 10.1073/pnas.68.11.2861. [DOI] [PMC free article] [PubMed] [Google Scholar]

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